Targeted therapy, the new frontier of cancer treatment, employs pharmacological tools (drugs or antibodies) specifically blocking crucial gene products that sustain the transformed phenotype. Currently, cancer targeted therapy is employed in the clinic for the treatment of chronic myelogenous leukemias (CML), addicted to the tyrosine kinase molecule ABL, for the treatment of a subset of Non-Small Cell Lung Cancers (NSCLC) and Colon-Rectum Carcinomas (CRC) relying on Epidermal Growth Factor Receptor (EGFR/HER-1) activation and for the treatment of BRAF-dependent melanomas.
Receptors with tyrosine kinase activity (RTKs) are interesting candidates for targeted therapy as they are often hyper-activated in several types of tumors. They can be inhibited by different types of targeting molecules, such as antibodies, that upon interaction with the extracellular part of the receptor are able to perturb the receptor-induced intracellular signaling, and chemically-synthesized small molecules that interfere with the receptor catalytic activity.
Among the different RTKs, the product of the c-met proto-oncogene, the Hepatocyte Growth Factor Receptor (HGFR/Met), is emerging as one of the most important activated oncogene in cancer. Met controls a genetic program known as ‘invasive growth’ that includes pro-mitogenic, pro-invasive and anti-apotoptic cues. Through these physiological signals, Met provides with a better fitness the tumor, helping it to overcome selective barriers in cancer progression. Moreover Met sustains tumor growth by its ability to promote tumor angiogenesis. In the last years, Met also resulted responsible for the aggressiveness developed by tumors treated with anti-angiogenic agents and for resistance to conventional radiotherapy. Additionally, MET gene alteration can be a primary cause of transformation, in all of those cases in which it has been genetically selected for the long term maintenance of the primary transformed phenotype.
All the above listed findings have prompted the development of several molecules suitable to inhibit Met signaling, including competitive inhibitors of HGF, chemical Met kinase inhibitors, anti-HGF and anti-Met antibodies. Some of these molecules, until now, have been tested only for research purpose. Clinical trials are currently ongoing with neutralizing anti-HGF antibodies, anti-Met antibodies and several small molecules.
From several view-points, an anti-Met antibody able to inhibit Met signaling would be preferable. Antibodies are highly specific, stable and, thank to their natural design, they are generally well tolerated by the host. In the last years, several efforts have been put to generate therapeutic anti-Met antibodies. However, a lot of failures have been registered, as the majority of the anti-Met antibodies behave as agonists, mimicking the HGF action. This is mostly due to the fact that, thanks to their bivalent structure, antibodies can stabilize receptor dimers, allowing trans-phosphorylation of Met, with its consequent activation. In one case, an agonist anti-Met antibody (5D5) has been engineered and converted in a monovalent form (One Armed-5D5) that, competing with HGF binding, is endowed with therapeutic potential (1,2). This molecule has been recently entered a phase III clinical trial for the treatment of a subset of Non Small Cell Lung Cancer patients, characterized by high level of Met expression in the tumors, in combination with erlotinib (3).
The monoclonal antibody DN30 is a mouse IgG2A directed against the extracellular moiety of the human Met receptor (4). It binds with sub-nanomolar affinity the fourth IPT domain of the Met receptor extracellular region. At the beginning, it was characterized as a partial agonist of Met, able to promote some, but not all, of the Met-mediated biological cell responses. Later it has been demonstrated that it can act as an inhibitor of tumor growth and metastasis through a mechanism of receptor ‘shedding’ (5). Receptor shedding is a physiologic cellular mechanism of protein degradation acting on diverse growth factors, cytokines, receptors and adhesion molecules. Met shedding is articulated in two steps: first a metalloprotease, the ADAM-10, cleaves the extracellular domain of Met recognizing a specific sequence localized immediately upstream to the trans-membrane region; then the remaining transmembrane fragment becomes substrate of a second protease (γ-secretase) that detaches the kinase-containing portion from the membrane and rapidly addresses it towards the proteasome degradation pathway (6,7). The enhancement of this mechanism exerted by the DN30 leads to a reduction in the number of Met receptors exposed at the cell surface. At the same time, it releases a soluble, ‘decoy’ ecto-domain in the extracellular space. The latter competes with the intact trans-membrane receptor for ligand binding and inhibits receptor homo-dimerization by forming hetero-dimeric complexes with bona fide Met. All these actions strongly impair Met-mediated signaling and result in prevention of the downstream biological effects.
Recently the present inventors demonstrated that the monovalent Fab fragment of the DN30 anti-Met monoclonal antibody (DN30 Fab) is cleared of any agonistic activity and maintains the ability to induce shedding, thus resulting in a potent Met inhibitor (8). Induction of Met shedding by DN30-Fab is dependent on the selective antibody-antigen interaction but is independent from receptor activation. This mechanism of action, based on the simple elimination of Met from the cell surface, gives to the DN30-Fab a strong advantage over other inhibitors, as it can be effective against all the forms of Met activation, whether HGF-dependent or not, induced by overexpression, mutation or gene amplification.
While the recombinant DN30-Fab is very attractive for clinical applications, the short Fab plasma half-life—mostly due to renal clearance—severely limits its use for patient treatment.
Currently, the most consolidated technique to improve the pharmacological properties of a Fab fragment is to increase its molecular weight by conjugation with Poly Ethylen Glycol (PEG). Fab PEGylation is a route pursued in most of the cases employing Fab in the clinic. The covalent attachment of the polymer chains to the antibody fragment, obtained efficiently and without loss of antigen binding properties, is not an obvious process and requires a strong effort of setting up.
Another technique used to improve the pharmacological properties of a Fab fragment is the one disclosed in EP-A-1 718 677. Such a procedure, used to generate the One Armed form of monoclonal antibody 5D5 commented above, is the production—on recombinant basis—of three different antibody chains in the same cell, the light chain (VL-CL), the heavy chain (VH-CH1-CH2-CH3) and the Fc portion of the heavy chain (CH2-CH3). The CH2-CH3 domains are not wild type: mutations, giving rise to specific tridimensional structures, are included. In one polypeptide, the CH2-CH3 region incorporates a sequence forming a protuberance, while in the other polypeptide the CH2-CH3 region contains a sequence forming a cavity, in which the protuberance can be inserted (Knob into hole structure). The presence of these tridimensional structures allows the preferable formation of heterodimers in which the heavy chain forms disulfide bonds with the Fc fragment, but does not exclude at all the formation of homodimers (i.e. two heavy chains linked together and two Fc linked together). Purification allowing the separation of the unwanted homodimers from the wanted heterodimers is mandatory. Thus the “One Armed procedure”, although very elegant, is cumbersome as it requires additional steps in the overall process that complicate the manufacturing and reduce the yield of the recombinant antibody.
It is therefore felt the necessity of a different solution to increase Fab plasma half-life for in vivo therapeutic use.